Project Summary Individuals with fair skin, who lack the ability to tan, are at increased risk for skin cancers including melanoma. This is not surprising given what is known about the photoprotective properties of the melanin pigment synthesized by melanocytes in the skin. Here we report that there is also a potentially dangerous side to pigmentation; electrophilic intermediates produced during the biosynthesis of melanin cause direct damage to DNA. We propose that melanin synthesis intermediates (MSIs) are mutagenic and could promote melanoma tumor formation. Unlike non-melanoma skin cancers, ultraviolet (UV) radiation signature mutations are not often found in the oncogenic drivers of melanomas, and melanomas often occur on skin that is rarely or never exposed to UV radiation. We hypothesize that chemically reactive MSIs such as dopaquinone covalently modify biomolecules including DNA and proteins, thereby causing mutations and disrupting the function of cellular proteins including the tumor suppressor p53, in a process that may initiate and/or promote tumorgenesis. We further propose that these processes can be suppressed by chemoprevention agents that detoxify MSIs. We will test this hypothesis in two Aims. In Aim 1 we will expand upon on our initial study (which showed that melanin synthesis damages plasmid DNA encoding luciferase) by performing a thorough kinetic characterization of this phenomenon. We will next determine the ability of human melanocytes to repair the damaged plasmid DNA in a host cell reactivation assay.The mutational spectrum of melanin- synthesis induced DNA damage will be characterized using the supF shuttle vector based mutagenesis assay and a state-of-the sequencing of plasmid DNA damaged by MSIs and replicated in mammalian cells. In Aim 2 we will examine the effects of MSIs on proteins We will first examine the effects of MSIs on known protein targets of intracellular electrophiles including p53 and the antioxidant selenoprotein thioredoxin reductase 1. We will identify additional proteins covalently modified by MSIs utilizing a biotinylated tyrosine analog (BTA) which labels proteins in melanoma cells after activation with the melanin synthesis enzyme tyrosinase. Melanocytes will be treated with BTA, and labeled proteins will be processed for identification by Western blot analysis of candidate proteins. We will identify novel protein targets of MSIs using a state-of-the art mass spectrometry-based proteomic analysis that is designed for the sensitive and specific identification of biotinylated proteins. The results of these studies will enhance understanding of the processes involved in initiation and promotion stages of melanoma tumor formation. This understanding will provide the basis for the rational design of new chemoprevention agents, identify candidate biomarkers of efficacy that can be used in animal studies and clinical trials of these new drugs, and enhance our ability to detect melanomas at the earliest stages when surgical excision is curative.